Tumors begin shedding tumor cells into the circulation at an early stage of cancer, typically prior to the appearance of clinical symptoms. In general, tumors with a diameter of about 1 mm are vascularized, which leads to as much as 4% of the tumor cells being shed into the circulation in a 24 hour period (Butler & Gullino, Cancer Res., vol. 35, pp. 512-516, 1975). These tumor cells are called circulating tumor cells (CTCs), and are generally, although not exclusively, epithelial cells shed from a solid tumor into the blood stream. CTCs are good indicators of the tumor from which they originated, which may be especially important for diagnosing early stage solid tumors which are usually too small to be detected by conventional methods such as mammography for breast cancer patients, or X-rays for lung cancer patients. Accordingly, detection of CTCs may, in some cases, be used as an early diagnostic tool for cancers, especially early stage cancers before the appearance of clinical symptoms.
However, the CTCs are only a very small fraction of the total cells in circulation. For example, for patients with carcinomas, it is estimated that about only one in ten million cells in the blood is a CTC. In addition, various types of CTCs differ significantly from each other depending on their origins, both in terms of morphology and their inclusion of cancer specific markers. For example, fibroblast-based tumor cells have a different morphology than breast cancer cells which arise from epithelial cells. Also, CTCs originating from breast cancer typically have markers such as CK+/DAPI+/CD45, while CTCs originating from pancreatic cancer typically have markers that include CK8+/CK19+. Therefore, differentiating CTCs originating from different cancers would require using different antibodies to target different markers that are specific for different cancers, optionally in combination with gathering information about cell morphology. These technical complexities make cancer diagnosis by detection of CTCs in a clinical setting very challenging.
An automated system for detecting, enumerating and/or characterizing CTCs in a blood sample employs immunomagnetic enrichment technology to target cancer specific cell surface markers. Another commercial technology for enumerating and/or characterizing CTCs is Fiber-optic Array Scanning Technology (FAST), which investigates nucleated cells from a blood sample as a monolayer of cells on a slide using a fluorescence-labelled antibody against a cancer specific cell surface marker to identify the CTCs on the slide. A third commercial technology is based on the microfluidic or “CTC-Chip” technique. Using breast cancer as an example, 1-3 mL of whole blood is directed to flow past 78,000 EpCam-coated microposts. EpCam+ cells will bind to the microposts and are subsequently stained with antibodies against CK, CD45, and DAPI, which are breast cancer specific cell surface markers.
Besides these commercial methods, other CTC detection methods have been proposed. U.S. Pat. No. 8,445,225 discloses a method for revealing, detecting, and characterizing CTCs in the blood of a patient. The method includes the steps of: a) obtaining a blood sample from a patient; b) removing or degrading a protein, carbohydrate, cell, or a combination thereof, in physical association with the surface of the CTCs present in the sample; and c) analyzing the CTCs revealed in step (b). Step (b) is used for exposing the cell surface of CTCs without causing damage to the CTCs themselves, thereby revealing the CTCs in the sample. The analysis of CTCs in step (c) involves characterizing the morphology of the CTCs via image analysis. The analysis may also include detecting cancer specific cell surface markers on the CTCs, which include EGFR, HER2, ERCC1, CXCR4, EpCAM, E-Cadherin, Mucin-1, Cytokeratin, PSA, PSMA, RRM1, Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1, cMET, EML4, and Leukocyte Associated Receptor (LAR).
U.S. Pat. No. 8,039,218 discloses a method of detecting CTCs in a body fluid from a patient. The method comprises obtaining the body fluid from the patient and detecting the expression of a panel of genes in the body fluid, where the expression of the panel of genes indicates the presence of CTCs in the body fluid. Genes useful for detecting melanoma cells includes GalNAc-T, MAGE-A3, MART-1, PAX-3, and TRP-2. Genes useful for detecting carcinoma cells include C-Met, MAGE-A3, Stanniocalcin-1, Stanniocalcin-2, mammaglobin, HSP27, GalNAc-T, CK20, and β-HCG.
WO 2014/126904 discloses a method for detecting CTCs using a labeled pituitary adenylate cyclase activating peptide (PACAP) or vasoactive intestinal peptide (VIP). The PACAP and VIP can both bind to the VPAC1 receptor and detect CTCs present in blood or urine, since the VPAC1 receptor is present on surface of CTCs from many different cancer types. The PACAP has the sequence: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gin-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys. The labeled peptide is said to be able to detect CTCs at a concentration of 5 cells/ml sample and correctly identify and distinguish CTCs from epithelial cells and white blood cells contained in the sample.
US 2012/0094275 discloses a highly sensitive assay which combines immunomagnetic enrichment with multiparameter flow cytometry or image cytometry to detect, enumerate and characterize CTCs in a blood sample. The assay uses ferrofluid with different antibodies incorporated therein for detecting CTCs originating from different types of cancer. The multiple antibodies present in the same ferrofluid do not appear to block or otherwise interfere with each other. Such ferrofluids are capable of binding specifically to CTCs of more than one type of cancer. The assay is especially useful to enable the capture of CTCs that have low EpCAM expression, but high expression of other cancer specific markers.
These known methods of detecting CTCs often require use of multiple antibodies to diagnose different types of cancers, without knowing beforehand the cancer type a subject may have. For example, in order to be able to detect a wide range of common cancers, the antibodies will have to include one or more that bind specifically to breast cancer markers consisting of MUC-1, estrogen, progesterone receptor, cathepsin D, p53, urokinase type plasminogen activator, epidermal growth factor, epidermal growth factor receptor, BRCA1, BRCA2, CA27.29, CA15.5, prostate specific antigen, plasminogen activator inhibitor and Her2-neu; one or more that bind specifically to prostate cancer markers consisting of prostate specific antigen, prostatic acid phosphatase, thymosin b-15, p53, HPC1 basic prostate gene, creatine kinase and prostate specific membrane antigen; one or more that bind specifically to colon cancer markers consisting of carcinoembryonic antigen, C protein, APC gene, p53 and matrix metalloproteinase (MMP-9); and one or more that bind specifically to bladder cancer markers consisting of nuclear matrix protein (NMP22), Bard Bladder tumor antigen (BTA), and fibrin degradation product (FDP). Use of such a large number of antibodies significantly increases the cost and rate of false diagnoses when these methods are used in a clinical setting.
The present invention provides a diagnostic method that encapsulates one or more CTCs before detection thereof. Cell encapsulation has been described in, for example, US 2014/0127290 which discloses a method of encapsulating living cells in microcapsules. In the method, the cells are suspended in a matrix within the microcapsules. The microcapsules include a core having living cells or cell aggregates suspended or encapsulated therein and a shell surrounding the core comprising a biocompatible hydrogel. US 2012/0231443 also discloses a method for encapsulating cells in a microcapsule, which has a diameter of less than about 100 μm. The microcapsule may be further coated with chitosan and alginate.
Encapsulation of cells is generally viewed as hindering the detection of the encapsulated cells, since encapsulation introduces an outer layer to the encapsulated cells which may make detection of the cells more difficult. The present invention provides a diagnostic method that involves detection of encapsulated CTCs. The CTCs are encapsulated in manner which renders them more suitable for binding to conditionally active antibodies (CABs).
This technique enables detection of CTCs originating from many different cancer types using a single antibody in a simple procedure. This diagnostic technique is applicable to many different types of cancers and can therefore significantly reduce the cost of screening and diagnosis of early stage cancers while at the same time reducing the number of false positives that result from the use of prior art methods.